Field of the Invention
The invention relates to a multidimensional liquid chromatography separation systems for protein separation. In particular, a multidimensional liquid chromatography separation systems using for protein online quick separation is related. The separation method for protein separation using this liquid chromatography separation system is also provided in the present invention.
Description of the Related Art
Liquid chromatography is the most effective methods for analysis, separation and purification of protein, which is widely used in the extraction and separation of active protein from body fluid of natural plant and animal, and preparation and production of all kinds of recombinant protein drug in genetic engineering, and is also the essential equipment in the biochemistry laboratory, for example, protein and peptide separation in proteomics research in order to find the “mark” protein. Because the environment composition biological macromolecules exist is very complicated, it is often called the separation and analysis of biological macromolecules (biopolymers) in complicated components. Chromatographic column is the core of the chromatographic analysis. Scientists have been trying to improve the effect of chromatographic column so that the separation and analysis of general more complex materials can be done on a sample by a single chromatographic column. Chromatographic column is the core of the chromatographic analysis.
For the separation of small solutes, the number of theoretical plate (NTP) of liquid chromatographic column is generally for 104-105. However, for the analysis of more complex mixture, such as component separation in cell, body fluids and tissue of animals, the resolution of conventionally chromatographic column by now is far less than demand, so two-dimensional liquid chromatography (2D-LC) or multidimensional liquid chromatography (mD-LC) must be used to partially solve these problems. The technology 2D-LC and mD-LC has become the research hot-point and development direction of the modern chromatography in future.
For 2D-LC and mD-LC separation, there are two ways: off-line and on-line. 2D-LC separation in early stage mainly employs off-line method. The first dimensional separation is collected by hands, and then the fraction is injected into the second dimensional chromatographic column for separation. If the collected fraction is directly injected into the second column, the sampling amount must be a very small part of the fraction. If whole or most of the collected fraction is required to inject into the second column, the collected fraction must be pre-treated (concentration, buffer exchange). Off-line method has shortcomings, such as time-consuming, difficult to operate, sample contamination easily, low recovery and poor repeatability etc.
To satisfy the request of 2D-LC and mD-LC methods, several companies have been exploring the on-line method accompanying with producing several liquid chromatographs. However, from the standpoint of principle, these chromatographs are only improved based on the conventional chromatograph, and a set of chromatographic column (2-3 pieces) are inserted in parallel manner, as well as switch interfaces between these parallel chromatographic columns assembled, so as to transporting the fraction from one of the chromatographic columns to another for the separation of next mode by on-line manner. There are many problems existing in these on-line 2D-LC and mD-LC methods, such as the problem of mobile phase compatibility should be solved between different patterns of chromatography combination and the required interface is also a key technical problem.
Switch interface is the key device to the whole system of mD-LC technique. Commonly used ones are sample loops storing and transferring samples alternately, parallel columns enriching analytic samples alternately, capture column enriching samples, as well integrated two chromatographic columns with no interface. However, these instruments all have fatal flaw. On-line 2D-LC and mD-LC liquid chromatograph with interfaces can only inject 1-10% of the samples (only a few microliter) collected in the first dimension to the second dimensional chromatographic separation. In addition, it can be not used for protein separation in preparation scale, especially in industrialization production scale; As for mixed-mode chromatographic column without interfaces, two kinds of mobile phases must be completely compatible. Only in some specific circumstances, such as strong cation exchange chromatography (SCX) packing material being the first dimension and reversed phase liquid chromatography (RPLC) packing material being the second dimension, can such on-line two-dimensional separation be done. If the packing materials are packed on the contrary, this no switcher method will be useless.
Based on the background of this scientific development, scientists put forward the method of “mixed-mode chromatography (MMC). MMC is a type of chromatographic method in which multiple interaction modes take place between the stationary phase and solutes in the feed. This method has higher selectivity and high chromatographic column load, which provides selections and ideas for the development of semi-preparation and the preparation chromatography. However, in comparing with the traditional chromatographic column with the separation mode controlled by only one kind of force, MMC is controlled mainly by one kind of the two forces and the other one is the auxiliary, thus it only plays a role of improving the main separation mode. As a result, it is still employed for “one column-one usefulness”.
As early as in the 80s, Guiochon et al. (Chromatographia, 17 (3), 124-121, 1983) have put forward the method of 2D-LC with two dimensional chromatographic columns. The two-dimensional here refers to is the two-dimensional (plane) chromatography in space and the two-dimensional chromatographic column is for square (10 cm×10 cm), just as the conventional thin-layer chromatography plate. They applied for a patent, but didn't get it promoted and it has not been found any application in literature. Generally speaking, what we use in two-dimensional chromatography is the cylindrical chromatographic column. Two years ago, the patent applicant et al. put forward the “on-line two-dimensional liquid chromatography of protein separation with a single-column”, using the cylindrical two-dimensional chromatographic column. Several valves and spiral sample loops are inserted into the conventional liquid chromatograph, and then, with a cylindrical two-dimensional chromatography column, we carried out the fast 2D-LC separation of proteins. However, there are still problems existing of this method: (1) The maximum flow rate of the chromatograph is limited to 5 mL/min and the maximum volume of the sample loop of the collect-reserve device is only 5 mL, its can only operate on the analysis scale and can be not applied to preparative and productive scales; (2) This instrument is set for protein separation with one chromatographic column, therefore, it can be not employed to approach to the goal to obtain target protein with high purity which can only be carried out by means of mD-LC method using several types of chromatographic columns (such as the purity of insulin injections for protein >99%); (3) There is no strong theoretical support to design a multi-dimensional chromatograph and can only manual operate relying on experience. We neither can design software which is necessary to control modern instruments. What's more, this method can not be applied for polypeptide separation at that time; (4) All the chromatographic columns are specially made for “2D-LC” and there is no commercial products, so it is difficult to be employed by other chromatographers; (5) More importantly, there are only several equipment accessories patching up disordered on the conventional liquid chromatograph which is only employed to support the possibility for establishing this method. There is no whole idea of the framework on manufacturing equipment, technical scheme or technical parameters on how this method can carry out. There are more advantages that the 2D-LC is implemented using cylindrical 2D chromatography columns (simply called it as 2D column) than the flat form 2D chromatography columns.
Even so, there are several potential advantages of this chromatography: All operations of 2D-LC analysis for protein, including the collection and storage of the fractions from the effluent of the first separation model, the on-line buffer exchange under a high flow rate, the re-equilibrium of the chromatographic system, the on-line re-injection of the samples back to the same 2D column to carry out the second dimensional separation, are all accomplished in a closed system, so as to realize the “on-line two-dimensional chromatogram rapid analysis for proteins with a single-column”. This system has features that not only can prevent from the environmental pollution, but also can make the target protein be quantitatively transported to the second dimension chromatographic separation. Therefore, the detection limit and sensitivity of low abundance functional protein in proteomics can be improved 10-100 times, which will greatly accelerate the speed of “top-down-mass spectrometry” strategy in proteomics. If using the MMC mixed mode column, as its column loading is much higher than conventional chromatographic column, it can greatly save cost in the production of protein drugs in large scale.
Considering the potential advantages of this method, the applicant explores an equipment using for protein separation “protein multidimensional liquid chromatography purification system”.